US9365826B2 - Cardiomyocyte medium with dialyzed serum - Google Patents

Cardiomyocyte medium with dialyzed serum Download PDF

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US9365826B2
US9365826B2 US13/164,461 US201113164461A US9365826B2 US 9365826 B2 US9365826 B2 US 9365826B2 US 201113164461 A US201113164461 A US 201113164461A US 9365826 B2 US9365826 B2 US 9365826B2
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medium
cardiomyocytes
serum
cell
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Nathan Meyer
Brad Swanson
Steve Fiene
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Fujifilm Cellular Dynamics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates generally to the field of cell culture. More particularly, it concerns cardiomyocyte maintenance and modulation.
  • a central challenge for research in regenerative medicine is to develop cell compositions that can help reconstitute cardiac function. It is estimated that nearly one in five men and women have some form of cardiovascular disease (National Health and Nutrition Examination Survey lil, 1988-94, Center of Disease Control and the American Heart Association). Widespread conditions include coronary heart disease (5% of the population), congenital cardiovascular defects (0.5%), and congestive heart failure (3%).
  • the pharmaceutical arts have produced small molecule drugs and biological compounds that can help limit the damage that occurs as a result of heart disease, but there is nothing commercially available to help regenerate the damaged tissue.
  • cardiomyocytes derived in vitro from pluripotent stem cells of various kinds, especially induced pluripotent stem cells.
  • a number of obstacles have stood in the way of developing a paradigm for maintaining homogeneity of cardiomyocyte lineage cells in culture.
  • aspects of the present invention overcome a major deficiency in the art by providing a method for maintaining the purity, particularly in terms of homogeneity, of cardiomyocyte populations.
  • a method for maintaining the purity of a population of purified cardiomyocytes comprising: culturing a purified population of cardiomyocytes in a medium used described below, wherein the population is cultured for at least one week to seven months and maintains the purity of cardiomyocytes.
  • cardiomyocytes comprising: culturing a population of cardiomyocytes in a medium described below, wherein the beating rate of the cardiomyocyte culture has enhanced stability relative to a medium containing serum that has not been treated to remove low molecular weight molecules.
  • Such a medium may be essentially free of serum; or contains serum, wherein the serum or medium has been treated to remove low molecular weight molecules (including low molecular weight growth factors).
  • the serum or the medium may be essentially free of low molecular weight growth factors or essentially free of low molecular weight molecules.
  • the low molecular weight molecules or growth factors may have a molecular weight of less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 kD or any intermediate size or size range.
  • the medium could contain dialyzed serum.
  • the serum could be dialyzed with a membrane with molecular weight cutoff of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 kD or any range derivable therefrom.
  • Certain aspects of the invention may also provide a composition comprising a cell population of cardiomyocytes and a medium.
  • the medium may contain serum, wherein said serum or medium has been treated to remove low molecular weight molecules, such as dialyzed serum.
  • the medium may comprise at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99% dialyzed serum or any intermediate ranges or numbers.
  • the medium may essentially free of serum or serum components.
  • the cell population may have at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9% cardiomyocytes or any intermediate range or numbers.
  • the population may comprise at least 90% or 95% cardiomyocytes.
  • the population may be essentially free of contaminating cells such as fibroblasts or undifferentiated pluripotent stem cells.
  • the cardiomyocytes may express at least one selectable or screenable transgene under the control of a cardiomyocyte-specific promoter.
  • the cardiomyocytes are mouse or human cardiomyocytes.
  • the cardiomyocytes may be cryopreserved cardiomyocytes.
  • the medium may comprise sugar or be essentially free of sugar.
  • the medium may comprise glucose or be essentially free of glucose.
  • the medium may comprise galactose, for example, about 1 to 20 mM or any range derivable therein.
  • the medium may further comprise pyruvate or pyruvic acid, such as about 0.1 to 10 mM pyruvate or pyruvic acid.
  • the medium may be a maintenance medium.
  • a maintenance medium may comprise galactose, pyruvate, and dialyzed serum, such as 10 mM galactose, 1 mM pyruvate and 10% dialyzed serum.
  • the cardiomyocytes used herein may be previously purified.
  • the purified population of cardiomyocytes may be essentially free of non-cardiomyocyte cells.
  • a cell population “essentially free” of non-cardiomyocyte cells refers to a cell population that contains up to or less than about 1%, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1, 0.01% (or any range derivable therein) non-cardiomyocyte cells, including a cell population that has 100% cardiomyocytes.
  • the invention involves a cardiomyocyte cell population that comprises at most or about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1% (or any range derivable therein) non-cardiomyocyte cells.
  • exemplary non-cardiomyocyte cells that tend to contaminate pure cardiomyocyte populations include fibroblasts, undifferentiated cells and other non-cardiomyocyte cells.
  • cardiomyocytes in the media as described herein maintains the purity of cardiomyocytes; it is contemplated that to achieve such advantages the cardiomyocytes as described herein may be cultured in the medium for at least or about 2, 3, 4, 5, 6 days, 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11 months, 1, 2, 3, 4, 5 years or any range derivable therein. Specifically, the population of cardiomyocytes may maintain the purify for at least seven months.
  • a method comprising: a) obtaining a first composition comprising a cell population of at least 90% cardiomyocytes and a medium that may contain serum, wherein said serum or medium has been treated to remove low molecular weight molecules, or a second composition comprising a second cell population of at least 90% cardiomyocytes and a medium essentially free of serum; and b) culturing said first or second composition.
  • the culturing may last for at least 8 hours, 16 hours, one, two, three, four, five, six or seven days or any intermediate numbers.
  • the first or second composition maintains the purity of cardiomyocytes in the culturing.
  • the method may further comprise contacting cardiomyocytes in the first or second composition with a compound and measuring beating frequency and/or field potential duration of the cardiomyocytes.
  • the compound may modulate the ion channel activity, beating frequency and/or field potential duration of the cardiomyocytes.
  • the compound that modulates beating frequency may be tetrodotoxin (Octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-[1,3]dioxocino[6,5-d]pyrimidine-4,7,10,11,12-pentol) or isoproterenol;
  • the compound that modulates field potential duration may be E-4031 ((1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonyl-aminobenzoyl)piperidine)) or terfenadine ((RS)-1-(4-tert-butylphenyl)-4- ⁇ 4-[hydroxy(diphenyl)methyl]piperidin-1-yl ⁇ -butan-1-ol).
  • the method may also comprise cryopreserving cardiomyocytes in the first or second composition prior to the culturing.
  • the method may also comprise purifying cardiomyocytes differentiated from a stem cell or trans-differentiated from a non-cardiomyocyte cell in vitro to obtain cardiomyocytes in the first or second composition.
  • “Maintaining” the cardiomyocyte purity refers to the purity of cardiomyocytes does not decrease significantly over time, preferably at most or about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% reduction in the cell purity or homogeneity of the cardiomyocyte population, or any range derivable therein. “Purity” of cardiomyocytes, as used herein, refers to the percentage of cardiomyocytes in a given culture condition. This could be determined by detection of cardiomyocytes based on the expression of endogenous cardiomyocyte marker or transgenic gene marker under the control of a cardiomyocyte-specific promoter.
  • the purity could be measured by quantifying the percentage of RFP positive cells in cardiomyocyte cultures derived from an iPS cell line that expresses RFP driven by a cardiomyocyte-specific promoter or by quantifying the percentage of cardiac troponin T positive cells in cardiomyocyte cultures
  • the present invention further includes a method of increasing heart beat rate as well as a method of decreasing heart rate oscillation (i.e., enhancing heart rate stability) by culturing cardiomyocyte populations in the media of the present invention.
  • a method for testing the effect of a compound on cardiomyocytes comprising: a) contacting cardiomyocytes with a compound, wherein the cardiomyocytes are cultured in a medium that (i) is essentially free of serum; or (ii) contains serum, wherein the serum or medium has been treated to remove low molecular weight molecules (including low molecular growth factors); and b) measuring beating frequency and/or field potential duration of the cardiomyocytes.
  • the method of measuring may comprise the use of multi-electrode array (MEA).
  • the cardiomyocytes described herein may be contacted with a compound that modulates cell viability or function in the medium as described herein.
  • a compound may modulate the ion channel activity, such as potassium channels or sodium channels.
  • the compound may modulate beating frequency, such as tetrodotoxin or isoproterenol, or modulate field potential duration, like E-4031 or terfenadine.
  • a composition comprising a population of cardiomyocytes and a medium.
  • the population of cardiomyocytes may comprise non-cardiomyocyte cells, such as fibroblasts or undifferentiated cells.
  • the medium may comprise galactose, pyruvate and be essentially free of serum.
  • the medium may comprise galactose, pyruvate and dialyzed serum.
  • the medium may comprise glucose or be essentially free of glucose.
  • the cardiomyocytes may be previously purified; for example, they comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or any intermediate range of non-cardiomyocytes or are essentially free of non-cardiomyocyte cells.
  • the cardiomyocyte population may be purified, isolated or enriched.
  • the purification, isolation or enrichment may comprise the use of endogenous or exogenous markers specifically expressed in cardiomyocytes.
  • the cardiomyocyte may express one or more selectable or screenable transgenes.
  • the transgene may be a selectable or screenable marker under a cardiomyocyte-specific promoter, such as a promoter of MYH6 (alpha myosin heavy chain) gene.
  • a marker may include an antigenic epitope, a fluorescent protein-encoding gene or an antibiotic resistance gene.
  • the population of cardiomyocytes may be isolated based on expression of the transgene.
  • the isolation of a cardiomyocyte population may be any method known in the art, such as fluorescence sorting or magnetic sorting.
  • the purity of cardiomyocytes may be based on the expression of transgene that specifically expresses in cardiomyocytes.
  • the population of cardiomyocytes may have been preserved prior to the culturing in the medium described above.
  • the cardiomyocytes may comprise human cardiomyocytes or cardiomyocytes from different sources, such as any other mammals, like primates, dogs, cats, or mice.
  • the cardiomyocytes may be directly isolated from a subject or may be differentiated from a stem cell or trans-differentiated from a non-cardiomyocyte cell in vitro.
  • a stem cell could be a pluripotent stem cell or a tissue stem cell, such as a cardiac stem cell or a non-cardiac lineage stem cell or progenitor cell.
  • the pluripotent stem cell may include an induced pluripotent stem cell, an embryonic stem cell, or a pluripotent stem cell derived by somatic cell nuclear transfer.
  • the medium may be essentially free of serum, or more particularly, chemically defined.
  • the medium contains serum but the serum or the medium has been treated to remove low molecular weight molecules, particularly low molecular weight growth factors in the serum.
  • the medium or the serum may be essentially free of low molecular weight molecules or may be essentially free of low molecular weight serum growth factors and other components.
  • the medium may contain glucose.
  • the glucose in the medium may be at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 ⁇ M or 1, 2, 3, 4, 5, 5.5, 6, 7, 8, 9, 10 mM.
  • the medium may be essentially free of glucose.
  • the medium may comprise a compound capable of forming a high energy phosphate bond, an acyl group carrier molecule, or a cardiomyocyte calcium channel modulator.
  • the medium may comprise creatine, carnitine, or taurine.
  • the medium may comprise insulin.
  • the medium may comprise galactose, fructose, mannose, sucrose, maltose, lactose, trehalose, turanose, pyruvate, pyruvic acid, glutamine, glutamic acid, aspartate, aspartic acid, lactate, lactic acid, or a combination thereof.
  • the medium may comprise galactose.
  • the galactose in the medium may be at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50 mM or any range derivable therein.
  • the medium may also comprise pyruvate or pyruvic acid.
  • the pyruvate or pyruvic acid in the medium may be at least or about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mM or any range derivable therein.
  • Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.
  • encode or “encoding” with reference to a nucleic acid are used to make the invention readily understandable by the skilled artisan; however, these terms may be used interchangeably with “comprise” or “comprising” respectively.
  • FIGS. 1A-1B Serum free glucose free medium maintains cardiomyocyte purity in vitro.
  • FIG. 1A Cardiomyocytes cultured in control medium (Cardiac Maintenance Medium: CMM) or serum free glucose free (SFGF) medium maintain similar cardiomyocyte viability when cultured for 14 days.
  • FIG. 1B Purified cardiomyocytes cultured in CMM demonstrate a loss of purity ( ⁇ 90%) after 14 days. Cardiomyocytes cultured in SFGF medium maintain a higher purity (>99%).
  • FIGS. 2A-2C DS-CMM medium maintains cardiomyocyte purity in vitro.
  • FIG. 2A Cardiomyocytes cultured in CMM or in glucose free medium supplemented with dialyzed serum, sodium pyruvate, and galactose (DS-CMM) maintain similar cardiomyocyte viability when cultured for 14 days.
  • FIG. 2B Purified cardiomyocytes cultured in CMM demonstrate a loss of purity ( ⁇ 90%) after 14 days. Cardiomyocytes cultured in the DS-CMM medium maintain a higher purity (>99%).
  • FIG. 2C Cryopreserved cardiomyocytes were cultured in DS-CMM for 210 days. Purity was assayed at thaw, 7 days, and 210 days with no significant change in purity.
  • FIG. 3 iCMM vs. SFGF vs. CMM.
  • Cells were seeded into CMM and transitioned to experimental medium at 24 hours. Cell counts and purity was assayed at 7 days. The SFGF medium and iCMM promote higher cardiomyocyte survival and purity after 7 days in culture.
  • FIG. 4 Dialyzed Serum inhibits the outgrowth of contaminating cells. Cardiomyocytes were cultured in DMEM, Normal Serum; DMEM-Glucose, Normal Serum; DMEM, Dialyzed Serum; or DS-CMM (also called iCMM). The two DMEM conditions prepared with dialyzed serum (DMEM, Dialyzed Serum and DS-CMM) maintained higher cardiomyocyte purity when compared to normal serum containing DMEM medium.
  • DMEM Dialyzed Serum
  • DS-CMM also called iCMM
  • FIGS. 5A-5B Glucose Concentration effects cardiomyocyte purity to a lesser extent.
  • FIG. 5A DMEM culture medium containing dialyzed serum but lacking glucose (iCMM, also known as DS-CMM) resulted in cultures with significantly higher cardiomyocyte purity compared to glucose containing DMEM culture media containing dialyzed serum.
  • FIG. 5B Lack of glucose in normal serum containing DMEM medium increases cardiomyocyte purity of the cell culture.
  • SFGF serum free glucose free
  • the invention is in part based on the finding that serum-free or dialyzed serum-containing media could be designed to retard the growth of non-cardiomyocyte cells in a cardiomyocyte population while maintaining a healthy cardiomyocyte population. Even within a substantially pure cardiomyocyte population, a low percentage of contaminating cells may be present. Under standard serum-containing culturing conditions, some proportion of contaminating cells could proliferate at a faster rate than the cardiomyocytes and may overtake the whole cell population. In some embodiments, media conditions of the present invention may also improve some functional properties of cardiomyocytes.
  • “Pluripotency” refers to a stem cell that has the potential to differentiate into all cells constituting one or more tissues or organs, for example, any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system).
  • endoderm internal stomach lining, gastrointestinal tract, the lungs
  • mesoderm muscle, bone, blood, urogenital
  • ectoderm epidermal tissues and nervous system.
  • “Pluripotent stem cells” used herein refer to cells that can differentiate into cells derived from any of the three germ layers, for example, descendants of totipotent cells or induced pluripotent stem cells.
  • iPS cells commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing or contacting reprogramming factors.
  • Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos.
  • Cardiomyocytes refers generally to any cardiomyocyte lineage cells, and can be taken to apply to cells at any stage of cardiomyocyte ontogeny without any restriction, unless otherwise specified.
  • cardiomyocytes may include both cardiomyocyte precursor cells and mature cardiomyocytes.
  • a “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” or “fragment,” which “encodes” a particular protein is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded.
  • a gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3′ to the gene sequence.
  • transgene refers to a gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means, such as an exogenous nucleic acid.
  • An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell.
  • an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
  • promoter is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3′ direction) coding sequence.
  • CMM or DMEM Cardiac DMEM with fetal bovine serum added Normal Serum Maintenance (5.5 mM glucose in total medium) Medium iCMM or DS- iCell Cardiac Modified DMEM without sodium CMM Maintenance pyruvate or glucose; dialyzed fetal Medium or bovine serum added; sodium pyruvate DMEM - and galactose added Glucose, ( ⁇ 0 mM Glucose in total medium) Dialyzed Serum DMEM - same Modified DMEM without sodium Glucose, Normal pyruvate or glucose; normal fetal bovine Serum serum added; sodium pyruvate and galactose added (0.55 mM glucose in total medium) DMEM, Dialyzed same DMEM with dialyzed fetal bovine Serum serum (4.95 mM glucose in total medium) FBS + Media same SFGF Medium with fetal bovine serum added
  • the culturing conditions according to certain aspects of the present invention could be appropriately defined depending on the medium used.
  • the medium can be prepared using a medium to be used for culturing animal cells as its basal medium.
  • the basal medium any of TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, ⁇ MEM, DMEM, Ham, RPMI 1640, and Fischer's media, as well as any combinations thereof, can be used, but the medium is not particularly limited thereto as far as it can be used for culturing animal cells.
  • the medium can be a serum-containing or serum-free medium.
  • the serum-free medium refers to a medium with essentially no serum or serum-derived components.
  • the serum-free medium may also be essentially free of blood-derived components or animal tissue-derived components (such as animal tissue-derived growth factors), especially growth factors or serum components with molecular weights up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 kD or any range derivable therefrom.
  • dialysis removes many small molecules from fetal bovine serum (FBS) such as glucose, salts and some non-protein bound serum molecules. This process may not remove hormones that are serum bound but it may reduce growth promotion capabilities for some cell types.
  • FBS fetal bovine serum
  • the dialysis process could be done by ultra filtration with a 10,000 molecular weight cut-off membrane.
  • the medium may contain or may not contain any alternatives to serum.
  • the alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, or equivalents thereto.
  • the alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example.
  • any commercially available materials can be used for more convenience.
  • the commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).
  • the medium may be chemically defined, which refers to a medium essentially free of animal-derived components.
  • the medium may comprise recombinant growth factors or proteins, for example, recombinant albumin.
  • the medium can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and inorganic salts.
  • concentration of 2-mercaptoethanol can be, for example, about 0.05 to 1.0 mM, and particularly about 0.1 to 0.5 mM, but the concentration is particularly not limited thereto as long as it is appropriate for culturing the cell(s).
  • a culture vessel used for culturing the cardiomyocytes can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, culture bag, and roller bottle, as long as it is capable of culturing the cardiomyocytes therein.
  • the cardiomyocytes may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, 2000 ml or any range derivable therein, depending on the needs of the culture.
  • the culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment.
  • the bioreactor may have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.
  • the culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose.
  • the cellular adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells.
  • the substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used).
  • the substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin and mixtures thereof for example MatrigelTM, and lysed cell membrane preparations (Klimanskaya et al., 2005).
  • the culturing temperature can be about 30 to 40° C., for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39° C. but particularly not limited to them.
  • the CO 2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein.
  • the oxygen tension can be at least or about 1, 5, 8, 10, 20%, or any range derivable therein.
  • Cardiomyocytes in certain aspects of the present invention could be prepared by differentiation of stem cells such as induced pluripotent stem cells or cardiac stem cells, or trans-differentiation of non-cardiac cells, including other tissue stem cells.
  • stem cells such as induced pluripotent stem cells or cardiac stem cells
  • trans-differentiation of non-cardiac cells including other tissue stem cells.
  • Differentiation of pluripotent stem cells can be induced in a variety of manners, such as in attached colonies or by formation of cell aggregates, e.g., in low-attachment environment, wherein those aggregates are referred to as embryoid bodies (EBs).
  • EBs embryoid bodies
  • the molecular and cellular morphogenic signals and events within EBs mimic many aspects of the natural ontogeny of such cells in a developing embryo.
  • cardiomyocytes could be prepared by direct differentiation of pluripotent stem cells.
  • Direct differentiation refers to a process for differentiating pluripotent stem cells into progeny that are enriched for cells of a particular tissue type without forming embryoid bodies (cell aggregates) as an intermediate. This may be done when the cells are plated on a solid substrate, although plating is not necessarily required if not explicitly specified. Direct differentiation is effected by culturing in a growth environment of media components, soluble factors, insoluble components in suspension or on the vessel wall, and other ingredients that accomplish the objective of directing the cells towards the desired tissue type.
  • Embryoid bodies are aggregates of cells derived from pluripotent stem cells, such as ES cells or iPS cells, and have been studied for years with mouse embryonic stem cells.
  • pluripotent stem cells such as ES cells or iPS cells
  • certain aspects of the invention may employ three-dimensional aggregates (i.e., embryoid bodies) as an intermediate step.
  • differentiation is initiated and the cells begin to a limited extent to recapitulate embryonic development. Though they cannot form trophectodermal tissue (which includes the placenta), cells of virtually every other type present in the organism can develop.
  • the present invention may further promote cardiac differentiation following aggregate formation.
  • Pluripotent stem cells may be seeded into aggregate promotion medium using any method known in the art of cell culture. For example, pluripotent stem cells may be seeded as a single colony or clonal group into aggregate promotion medium, and pluripotent stem cells may also be seeded as essentially individual cells. In some embodiments, pluripotent stem cells are dissociated into essentially individual cells using mechanical or enzymatic methods known in the art.
  • pluripotent stem cells may be exposed to a proteolytic enzyme which disrupts the connections between cells and the culturing surface and between the cells themselves.
  • Enzymes which may be used to individualize pluripotent stem cells for aggregate formation and differentiation may include, but are not limited to, trypsin, in its various commercial formulations, such as TrypLE, or a mixture of enzymes such as Accutase®.
  • pluripotent cells may be added or seeded as essentially individual (or dispersed) cells to a culturing medium for culture formation on a culture surface.
  • the culturing medium into which cells are seeded may comprise TeSR medium or mTeSR medium and a survival factor.
  • dispersed pluripotent cells are seeded into a culturing medium at a density of from about 10 4 cells/ml to about 10 10 cells/ml. More particularly, pluripotent cells are seeded at a density of from about 10 5 cells/ml to about 10 7 cells/ml, or about 0.5 ⁇ 10 6 cells/ml to about 3 ⁇ 10 6 cells/ml.
  • a culturing surface may be comprised of essentially any material which is compatible with standard aseptic cell culture methods in the art, for example, a non-adherent surface.
  • a culturing surface may additionally comprise a matrix component as described herein.
  • a matrix component may be applied to a culturing surface before contacting the surface with cells and medium.
  • Cardiomyocyte lineage cells can be obtained from undifferentiated stem cells by culturing or differentiating in a special growth environment that enriches for cells with the desired phenotype (either by outgrowth of the desired cells, or by inhibition or killing of other cell types).
  • the iPS cells may be differentiated into cardiac cells incorporating the disclosed methods. Differentiation can be initiated by forming embryoid bodies or aggregates as described above: for example, by overgrowth of a pluripotent stem cell culture, or by culturing pluripotent stem cells in suspension in culture vessels having a substrate with low adhesion properties which allows EB formation. Pluripotent stem cells could be harvested by brief collagenase digestion, dissociated into clusters, and plated in non-adherent cell culture plates (WO 01/51616; U.S. Pat. No. 6,602,711, incorporated by reference).
  • the EBs can be produced encapsulated in alginate or other suitable nutrient-permeable matrix, which may help improve the uniformity of EB diameter and consistency of the cells produced (WO 03/004626, Zandstra et al., incorporated by reference).
  • a medium that contains serum or serum equivalent promotes foci of contracting cells of the cardiomyocyte lineage: for example, about 20% fetal bovine serum, or a serum supplement such as B27 or N2 in a suitable growth medium such as RPMI.
  • More exemplary methods of cardiac differentiation may include embryoid body (EB) methods (Zhang et al., 2009, which is incorporated by reference), OP9 stroma cell methods (Narazaki, et al., 2008, which is incorporated by reference), or growth factor/chemical methods (see U.S. Patent Publication Nos. 20080038820, 20080226558, 20080254003 and 20090047739, all incorporated herein by reference in their entirety).
  • EB embryoid body
  • the cells can be cultured with factors and factor combinations that enhance proliferation or survival of cardiomyocyte type cells, or inhibit the growth of other cell types.
  • the effect may be due to a direct effect on the cell itself, or due to an effect on another cell type, which in turn enhances cardiomyocyte formation.
  • induction medium for cardiac differentiation may include, but is not limited to, precardiac explants, precardiac mesoderm conditioned medium, and mesoderm secreted growth factors such as HGF.
  • Additional factors thought to induce differentiation of pluripotent stem cells into cells of the mesoderm layer, or facilitate further differentiation into cardiomyocyte lineage cells include the following:
  • TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3 and other members of the TGF- ⁇ superfamily illustrated below TGF- ⁇ receptor activate Type I and Type II serine kinases and cause phosphorylation of the SMAD effector.
  • Morphogens like Activin A and Activin B members of the TGF- ⁇ superfamily.
  • Insulin-like growth factors such as IGF I and IGF II.
  • Bone morphogenic proteins members of the TGF- ⁇ superfamily, exemplified by BMP-2 and BMP-4.
  • Fibroblast growth factors (exemplified by bFGF, FGF-4, and FGF-8), other ligands that activate cytosolic kinase raf-1 and mitogen-activated proteins kinase (MAPK), and other mitogens such as epidermal growth factor (EGF).
  • bFGF cytosolic kinase raf-1 and mitogen-activated proteins kinase
  • MAPK mitogen-activated proteins kinase
  • EGF epidermal growth factor
  • Nucleotide analogs that affect DNA methylation and altering expression of cardiomyocyte-related genes e.g., 5-aza-deoxy-cytidine.
  • Exemplary effective combinations of cardiotropic agents include use of a morphogen like Activin A and a plurality of growth factors, such as those included in the TGF- ⁇ and IGF families during the early commitment stage, optionally supplemented with additional cardiotropins such as one or more fibroblast growth factors, bone morphogenic proteins, and platelet-derived growth factors.
  • a morphogen like Activin A and a plurality of growth factors such as those included in the TGF- ⁇ and IGF families during the early commitment stage, optionally supplemented with additional cardiotropins such as one or more fibroblast growth factors, bone morphogenic proteins, and platelet-derived growth factors.
  • IGF II insulin-like growth factor II
  • Wnt proteins normally stabilize and cause nuclear translocation of a cytoplasmic molecule, ⁇ -catenin, which comprises a portion of the transcription factor TCF. This changes transcriptional activity of multiple genes.
  • ⁇ -catenin is phosphorylated by the kinase GSK3 ⁇ , which both destabilizes ⁇ -catenin and keeps it in the cytoplasm.
  • Wnt activators like IGF II apparently limit cardiomyocyte differentiation
  • certain aspects of this invention may include culturing with Wnt antagonists to increase the extent or proportion of cardiomyocyte differentiation of pluripotent stem cells.
  • Wnt signaling can be inhibited by injection of synthetic mRNA encoding either DKK-1 or Crescent (secreted proteins that bind and inactivate Wnts) (Schneider et al., 2001), or by infection with a retrovirus encoding DKK-1 (Marvin et al., 2001).
  • the Wnt pathway can be inhibited by increasing the activity of the kinase GSK3 ⁇ , for example, by culturing the cells with factors such as IL-6 or glucocorticoids.
  • FGF or a combination of FGF and HGF are used to culture pluripotent stem cells, cell aggregates, or differentiated stem cells, which may promote cardiac induction of stem cells.
  • FGF may be added at a concentration of at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/ml or any range derivable therein.
  • HGF hepatic growth factor
  • HGF hepatic growth factor
  • cardiomyocytes could be derived from pluripotent stem cells in vitro.
  • pluripotent stem cell refers to a cell capable of giving rise to cells of all three germinal layers, that is, endoderm, mesoderm and ectoderm. Although in theory a pluripotent stem cell can differentiate into any cell of the body, the experimental determination of pluripotency is typically based on differentiation of a pluripotent cell into several cell types of each germinal layer.
  • a pluripotent stem cell is an embryonic stem (ES) cell derived from the inner cell mass of a blastocyst.
  • the pluripotent stem cell is an induced pluripotent stem cell derived by reprogramming somatic cells.
  • the pluripotent stem cell is an embryonic stem cell derived by somatic cell nuclear transfer.
  • Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a blastocyst.
  • ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo, then culturing the inner mass cells on a feeder layer of non-growing cells. Under appropriate conditions, colonies of proliferating, undifferentiated ES cells are produced. The colonies can be removed, dissociated into individual cells, then replated on a fresh feeder layer. The replated cells can continue to proliferate, producing new colonies of undifferentiated ES cells. The new colonies can then be removed, dissociated, replated again and allowed to grow.
  • a “primary cell culture” is a culture of cells directly obtained from a tissue such as the inner cell mass of a blastocyst.
  • a “subculture” is any culture derived from the primary cell culture.
  • mouse ES cells Methods for obtaining mouse ES cells are well known.
  • a preimplantation blastocyst from the 129 strain of mice is treated with mouse antiserum to remove the trophoectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts in medium containing fetal calf serum. Colonies of undifferentiated ES cells that develop are subcultured on mouse embryonic fibroblast feeder layers in the presence of fetal calf serum to produce populations of ES cells.
  • mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture medium (Smith, 2000).
  • LIF cytokine leukemia inhibitory factor
  • mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).
  • Human ES cells can be obtained from blastocysts using previously described methods (Thomson et al., 1995; Thomson and Marshall, 1998; Reubinoff et al, 2000.) In one method, day-5 human blastocysts are exposed to rabbit anti-human spleen cell antiserum, then exposed to a 1:5 dilution of Guinea pig complement to lyse trophectoderm cells. After removing the lysed trophectoderm cells from the intact inner cell mass, the inner cell mass is cultured on a feeder layer of gamma-inactivated mouse embryonic fibroblasts and in the presence of fetal bovine serum.
  • clumps of cells derived from the inner cell mass can be chemically (i.e. exposed to trypsin) or mechanically dissociated and replated in fresh medium containing fetal bovine serum and a feeder layer of mouse embryonic fibroblasts.
  • colonies having undifferentiated morphology are selected by micropipette, mechanically dissociated into clumps, and replated (see U.S. Pat. No. 6,833,269).
  • ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells can be routinely passaged by brief trypsinization or by selection of individual colonies by micropipette.
  • human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000).
  • human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MatrigelTM or laminin in the presence of “conditioned” medium containing basic fibroblast growth factor (Xu et al., 2001). The medium is previously conditioned by coculturing with fibroblasts.
  • ES cell lines Another source of ES cells are established ES cell lines.
  • Various mouse cell lines and human ES cell lines are known and conditions for their growth and propagation have been defined.
  • the mouse CGR8 cell line was established from the inner cell mass of mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without feeder layers.
  • human ES cell lines H1, H7, H9, H13 and H14 were established by Thompson et al.
  • subclones H9.1 and H9.2 of the H9 line have been developed. It is anticipated that virtually any ES or stem cell line known in the art and may be used with the present invention, such as, e.g., those described in Yu and Thompson, 2008, which is incorporated herein by reference.
  • the source of ES cells for use in connection with the present invention can be a blastocyst, cells derived from culturing the inner cell mass of a blastocyst, or cells obtained from cultures of established cell lines.
  • ES cells can refer to inner cell mass cells of a blastocyst, ES cells obtained from cultures of inner mass cells, and ES cells obtained from cultures of ES cell lines.
  • Induced pluripotent stem (iPS) cells are cells which have the characteristics of ES cells but are obtained by the reprogramming of differentiated somatic cells. Induced pluripotent stem cells have been obtained by various methods. In one method, adult human dermal fibroblasts are transfected with transcription factors Oct4, Sox2, c-Myc and Klf4 using retroviral transduction (Takahashi et al., 2007). The transfected cells are plated on SNL feeder cells (a mouse cell fibroblast cell line that produces LIF) in medium supplemented with basic fibroblast growth factor (bFGF). After approximately 25 days, colonies resembling human ES cell colonies appear in culture. The ES cell-like colonies are picked and expanded on feeder cells in the presence of bFGF.
  • SNL feeder cells a mouse cell fibroblast cell line that produces LIF
  • bFGF basic fibroblast growth factor
  • cells of the ES cell-like colonies are induced pluripotent stem cells.
  • the induced pluripotent stem cells are morphologically similar to human ES cells, and express various human ES cell markers. Also, when grown under conditions that are known to result in differentiation of human ES cells, the induced pluripotent stem cells differentiate accordingly.
  • the induced pluripotent stem cells can differentiate into cells having neuronal structures and neuronal markers. It is anticipated that virtually any iPS cells or cell lines may be used with the present invention, including, e.g., those described in Yu and Thompson, 2008.
  • human fetal or newborn fibroblasts are transfected with four genes, Oct4, Sox2, Nanog and Lin28 using lentivirus transduction (Yu et al., 2007).
  • colonies with human ES cell morphology become visible.
  • the colonies are picked and expanded.
  • the induced pluripotent stem cells making up the colonies are morphologically similar to human ES cells, express various human ES cell markers, and form teratomas having neural tissue, cartilage and gut epithelium after injection into mice.
  • Sox may be Sox-1, Sox-2, Sox-3, Sox-15, or Sox-18; Oct may be Oct-4.
  • Additional factors may increase the reprogramming efficiency, like Nanog, Lin28, Klf4, or c-Myc; specific sets of reprogramming factors may be a set comprising Sox-2, Oct-4, Nanog and, optionally, Lin-28; or comprising Sox-2, Oct4, Klf and, optionally, c-Myc.
  • IPS cells like ES cells, have characteristic antigens that can be identified or confirmed by immunohistochemistry or flow cytometry, using antibodies for SSEA-1, SSEA-3 and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda Md.), and TRA-1-60 and TRA-1-81 (Andrews et al., 1987). Pluripotency of embryonic stem cells can be confirmed by injecting approximately 0.5-10 ⁇ 10 6 cells into the rear leg muscles of 8-12 week old male SCID mice. Teratomas develop that demonstrate at least one cell type of each of the three germ layers.
  • iPS cells are made from reprogramming somatic cells using reprogramming factors comprising Oct family member and a Sox family member, such as Oct4 and Sox2 in combination with Klf or Nanog as describe above.
  • the somatic cell in the present invention may be any somatic cell that can be induced to pluripotency, such as a fibroblast, a keratinocyte, a hematopoietic cell, a mesenchymal cell, a liver cell, a stomach cell, or a ⁇ cell.
  • T cells may also be used as source of somatic cells for reprogramming (see U.S. Application No. 61/184,546, incorporated herein by reference).
  • Reprogramming factors may be expressed from expression cassettes comprised in one or more vectors, such as an integrating vector or an episomal vector, such as a EBV element-based system (see U.S. application Ser. No. 12/478,154, incorporated herein by reference; Yu et al., 2009).
  • reprogramming proteins could be introduced directly into somatic cells by protein transduction (see U.S. application Ser. No. 12/723,063, incorporated herein by reference) or RNA transfection (see U.S. application Ser. No. 12/735,060).
  • Pluripotent stem cells can be prepared by means of somatic cell nuclear transfer, in which a donor nucleus is transferred into a spindle-free oocyte. Stem cells produced by nuclear transfer are genetically identical to the donor nuclei.
  • donor fibroblast nuclei from skin fibroblasts of a rhesus macaque are introduced into the cytoplasm of spindle-free, mature metaphase II rhesus macaque ooctyes by electrofusion (Byrne et al., 2007).
  • the fused oocytes are activated by exposure to ionomycin, then incubated until the blastocyst stage.
  • ES cells refers to embryonic stem cells derived from embryos containing fertilized nuclei. ES cells are distinguished from embryonic stem cells produced by nuclear transfer, which are referred to as “embryonic stem cells derived by somatic cell nuclear transfer.”
  • the cardiomyocyte can be characterized according to a number of phenotypic criteria, for example, the cardiomyocytes can purified or isolated, or alternatively, the purity of cardiomyocytes in a culture can be determined based on detection of cardiomyocytes in the culture.
  • Cardiomyocytes derived from stem cells such as pluripotent stem cell, have morphological characteristics of cardiomyocytes from other sources. They can be spindle, round, triangular or multi-angular shaped, and they may show striations characteristic of sarcomeric structures detectable by immunostaining. They may form flattened sheets of cells, or aggregates that stay attached to the substrate or float in suspension, showing typical sarcomeres and atrial granules when examined by electron microscopy.
  • the purity of cardiomyocytes may be determined by detecting cardiomyocytes which express an exogenous marker gene under the control of a promoter of a cardiomyocyte-specific marker or which expression an endogenous cardiomyocyte-specific marker.
  • detection may be used to isolate or purify the cardiomyocytes for a long-term storage in a medium described in certain aspects of the invention.
  • the cardiomyocyte-specific markers may include:
  • cTnI Cardiac troponin I
  • Nkx2.5 a cardiac transcription factor expressed in cardiac mesoderm during early mouse embryonic development, which persists in the developing heart.
  • Atrial natriuretic factor a hormone expressed in developing heart and fetal cardiomyocytes but down-regulated in adults. It is considered a good marker for cardiomyocytes because it is expressed in a highly specific manner in cardiac cells but not skeletal myocytes.
  • MHC Myosin heavy chain
  • GATA-4 a transcription factor that is highly expressed in cardiac mesoderm and persists in the developing heart. It regulates many cardiac genes and plays a role in cardiogenesis
  • MEF-2A MEF-2B, MEF-2C, MEF-2D; transcription factors that are expressed in cardiac mesoderm and persist in developing heart
  • N-cadherin which mediates adhesion among cardiac cells
  • Connexin 43 which forms the gap junction between cardiomyocytes.
  • CK-MB creatine kinase MB
  • myoglobin which are elevated in serum following myocardial infarction
  • Cardiomyocyte-specific markers can be detected using any suitable immunological technique—such as flow immunocytometry or affinity adsorption for cell-surface markers, immunocytochemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers, Western blot analysis of cellular extracts, and enzyme-linked immunoassay, for cellular extracts or products secreted into the medium.
  • immunological technique such as flow immunocytometry or affinity adsorption for cell-surface markers, immunocytochemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers, Western blot analysis of cellular extracts, and enzyme-linked immunoassay, for cellular extracts or products secreted into the medium.
  • Antibodies that distinguish cardiac markers like cTnI and cTnT from other isoforms are available commercially from suppliers like Sigma and Spectral Diagnostics.
  • Expression of an antigen by a cell is said to be antibody-detectable if a significantly detectable amount of antibody will bind to the antigen in a standard immunocytochemistry or flow cytometry assay, optionally after fixation of the cells, and optionally using a labeled secondary antibody.
  • cardiomyocyte-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods using publicly available sequence data (GenBank).
  • RT-PCR reverse transcriptase initiated polymerase chain reaction
  • tissue-specific markers as detected at the protein or mRNA level is considered positive if the level is at least or about 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-fold, and more particularly more than 10-, 20-, 30, 40-, or 50-fold above that of a control cell, such as an undifferentiated pluripotent stem cell or other unrelated cell type.
  • markers Once markers have been identified on the surface of cells of the desired phenotype, they can be used for immunoselection to further enrich the population by techniques such as immunopanning or antibody-mediated fluorescence-activated cell sorting.
  • pluripotent stem cell-derived cardiomyocytes often show spontaneous periodic contractile activity. This means that when they are cultured in a suitable tissue culture environment with an appropriate Ca 2+ concentration and electrolyte balance, the cells can be observed to contract across one axis of the cell, and then release from contraction, without having to add any additional components to the culture medium.
  • the contractions are periodic, which means that they repeat on a regular or irregular basis, at a frequency between about 6 and 200 contractions per minute, and often between about 20 and about 90 contractions per minute in normal buffer.
  • Individual cells may show spontaneous periodic contractile activity on their own, or they may show spontaneous periodic contractile activity in concert with neighboring cells in a tissue, cell aggregate, or cultured cell mass.
  • the contractile activity of the cells can be characterized according to the influence of culture conditions on the nature and frequency of contractions.
  • Compounds that reduce available Ca 2+ concentration or otherwise interfere with transmembrane transport of Ca 2+ often affect contractile activity.
  • the L-type calcium channel blocker diltiazem inhibits contractile activity in a dose-dependent manner.
  • adrenoceptor agonists like isoprenaline and phenylephrine have a positive chronotropic effect.
  • Further characterization of functional properties of the cell can involve characterizing channels for Na + , K + , and Ca 2+ . Electrophysiology can be studied by patch clamp analysis for cardiomyocyte like action potentials.
  • Functional attributes provide a manner of characterizing cells and their precursors in vitro, but may not be necessary for some of the uses referred to in this disclosure.
  • a mixed cell population enriched for cells bearing some of the markers listed above, but not all of the functional or electrophysiology properties can be of considerable therapeutic benefit if they are capable of grafting to impaired cardiac tissue, and acquiring in vivo the functional properties needed to supplement cardiac function.
  • the cells of this invention can be made to contain one or more genetic alterations by genetic engineering of the cells either before or after differentiation (US 2002/0168766).
  • a cell is said to be “genetically altered” or “transgenic” when a polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide.
  • the cells can be processed to increase their replication potential by genetically altering the cells to express telomerase reverse transcriptase, either before or after they progress to restricted developmental lineage cells or terminally differentiated cells (US 2003/0022367).
  • the cells of this invention can also be genetically altered in order to enhance their ability to be involved in tissue regeneration, or to deliver a therapeutic gene to a site of administration.
  • a vector is designed using the known encoding sequence for the desired gene, operatively linked to a promoter that is either pan-specific or specifically active in the differentiated cell type.
  • cells that are genetically altered to express one or more growth factors of various types such as FGF, cardiotropic factors such as atrial natriuretic factor, cripto, and cardiac transcription regulation factors, such as GATA-4, Nkx2.5, and MEF2-C. Production of these factors at the site of administration may facilitate adoption of the functional phenotype, enhance the beneficial effect of the administered cell, or increase proliferation or activity of host cells neighboring the treatment site.
  • cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in an expression vector, such as a selectable or screenable marker.
  • a marker in an expression vector such as a selectable or screenable marker.
  • Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector, or help enrich or identify differentiated cardiac cells by using a cardiomyocyte-specific promoter, such as promoters of cardiac troponin I (cTnI), cardiac troponin T (cTnT), ⁇ -myosin heavy chain (MYH6), myosin light chain-2v (MLC-2v), GATA-4, Nkx2.5, N-cadherin, ⁇ 1-adrenoceptor, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF).
  • CK-MB creatine kinase MB
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, blasticidin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions are also contemplated.
  • screenable markers such as GFP, whose basis is colorimetric analysis
  • Examples of such screenable include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants and enzymes (e.g., ⁇ -galactosidase).
  • cell surface proteins e.g., CD4, HA epitope
  • fluorescent proteins e.g., CD4, HA epitope
  • enzymes e.g., ⁇ -galactosidase
  • the vector containing cells may be isolated, e.g., by FACS using fluorescently-tagged antibodies to the cell surface protein or substrates that can be converted to fluorescent products by a vector encoded enzyme.
  • the screenable marker encodes a fluorescent protein.
  • a broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum (see Table 1 for non-limiting examples). Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata , and reef corals belonging to the class Anthozoa.
  • screenable enzymes such as chloramphenicol acetyltransferase (CAT) may be utilized.
  • CAT chloramphenicol acetyltransferase
  • One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.
  • Certain aspects of this invention provide a method to culture cells of the cardiomyocyte lineage.
  • the culture method or medium of the present invention could affect functional properties of the cardiomyocytes.
  • the culture medium could facilitate beating rate or reduce the incidence of beating rate oscillations.
  • Such cultured cardiomyocytes may be used for a number of important research, development, and commercial purposes.
  • Cardiomyocytes of this invention can be used commercially to screen for factors (such as solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of such cells and their various progeny, such as beating frequency or beating rate oscillations.
  • factors such as solvents, small molecule drugs, peptides, oligonucleotides
  • environmental conditions such as culture conditions or manipulation
  • the culture medium as described above may increase beating frequency and reduce the incidence of beating rate oscillations, thereby increasing the stability of beating frequency recordings.
  • the cardiomyocytes could be cultured in the medium as described above to facilitate a) normal beating frequency of cardiomyocytes; b) normal field potential duration of beating cardiomyocytes; c) beating frequency of cardiomyocytes treated with compounds that may effect beating frequency; or d) field potential duration of cardiomyocytes treated with compounds that may effect field potential duration.
  • Beating (contractile) frequency of cardiomyocytes can be modulated by culture media pH, temperature, or a modulator drug.
  • exemplary non-limiting modulator drugs include catecholamine, a calcium channel blocker, or potassium.
  • Cardiomyocytes as well as populations of cardiomyocytes including enriched or selected cardiomyocytes of any developmental, maturation or differentiation stage thereof can be used to screen for or identify cardioactive agents.
  • a cardiomyocyte population used in a screen or identification method includes nodal, sino-atrial or pacemaker cells, mature contractile cardiomyocytes, immature cardiomyocytes (cardioblasts), or a mixed population thereof.
  • Effect of cell function can be assessed using any standard assay to observe phenotype or activity of cardiomyocytes, such as marker expression, receptor binding, contractile activity, or electrophysiology—either in cell culture or in vivo. Pharmaceutical candidates can also be tested for their effect on contractile activity—such as whether they increase or decrease the extent or frequency of contraction. Where an effect is observed, the concentration of the compound can be titrated to determine the median effective dose (ED 50 ).
  • ED 50 median effective dose
  • cardiomyocytes cultured in the medium according to certain aspects of the invention could be used to measure functional properties of the cardiomyocytes, particularly cardiac specific electrical activity, such as beating frequency or field potential.
  • Detection of cardiac specific electrical activity of the cells and tissues of the present invention may be effected by monitoring the electrical activity thereof via a multielectrode array.
  • Suitable multielectrode arrays may be obtained from Multi Channel Systems, Reutlingen, Germany.
  • the multielectrode array could be a two-dimensional orthogonal array which includes 60 or more electrodes positioned 100 ⁇ m or less apart.
  • the multielectrode array is configured to obtain data characterizing cardiac specific electrical activity with a frequency greater than a range selected from 1-25 kHz.
  • Monitoring electrical activity in the cells and tissues of the present invention can be used to provide many different types of important and novel information regarding electrical activity of cells and tissues of the present invention.
  • such monitoring can be used to monitor electrical activity individually at each electrode, or more advantageously, such monitoring can be used to generate electrical activity propagation maps, also termed herein “activation maps”, depicting electrical activity as a function of local activation time at each electrode, for example in the form of a color-coded gradient.
  • activation maps can be used to depict conduction velocity and conduction directionality of propagative electrical activity, preferably in the form of conduction velocity vectors, of electrical activity propagation over an area of the microelectrode array,
  • a method includes contacting a cardiomyocyte with a test agent; and determining if the test agent modulates an activity or function of cardiomyocytes within the population.
  • a test agent modulating an activity or function of cardiomyocytes within the population identifies the test agent as a cardioactive agent.
  • Exemplary activity or function that can be modulated include contraction or beating, or production of a metabolic product (e.g., production of one or more of urea, creatine or CO2), or intracellular enzyme (e.g., one or more of lactate dehydrogenase, creatine phosphokinase (CPK), creatine kinase (CK) or troponin), or cellular apoptosis, necrosis, death; or de-differentiation, maturation, or division.
  • a metabolic product e.g., production of one or more of urea, creatine or CO2
  • intracellular enzyme e.g., one or more of lactate dehydrogenase, creatine phosphokinase (CPK), creatine kinase (CK) or troponin
  • CPK creatine phosphokinase
  • CK creatine kinase
  • troponin e.g., cellular apoptosis, necrosis, death; or de-different
  • Methods of screening and identifying cardioactive agents include those suitable for high throughput screening, which include arrays of cardiomyocyte cells (e.g., microarrays) positioned or placed, optionally at pre-determined locations or addresses.
  • High-throughput robotic or manual handling methods can probe chemical interactions and determine levels of expression of many genes in a short period of time. Techniques have been developed that utilize molecular signals (e.g., fluorophores) and automated analyses that process information at a very rapid rate (see, e.g., Pinhasov et al., 2004). For example, microarray technology has been extensively utilized to probe the interactions of thousands of genes at once, while providing information for specific genes (see, e.g., Mocellin and Rossi, 2007).
  • cardiomyocyte cells e.g., cardiomyoblasts, cardiomyocytes or sino-atrial nodal cells
  • a culture dish, tube, flask, roller bottle or plate e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish
  • Libraries that can be screened include, for example, small molecule libraries, siRNA libraries, and adenoviral transfection vectors.
  • cardiomyocyte cells e.g., cardiomyoblasts, cardiomyocytes or sino-atrial nodal cells
  • a culture dish, tube, flask, roller bottle or plate e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish
  • small molecule libraries, siRNA libraries, adenoviral transfection vectors, and gene based microarray approaches can identify various therapeutic and cardiac liability targets.
  • Such techniques also allow direct high-throughput measurement of cardiac intervention strategies by means of fluorescent reporter dyes and biomarkers for cell health and morphological phenotype, expression of fluorescent reporter proteins, various FRET approaches and direct measurement of electrophysiological currents in live cells.
  • a defined, serum free, glucose free (SFGF) medium was designed to maintain the survival of cardiomyocyte cells grown in vitro.
  • the medium has also been designed to maintain the purity cardiomyocyte cells grown in vitro.
  • Culture medium with standard levels of glucose and normal serum induce outgrowth of the non-cardiomyocyte population. This medium maintains the cardiomyocyte purity of the culture for at least 2 weeks.
  • the serum free glucose free (SFGF) medium contains:
  • Creatine stock solution Sigma Cat. 27890-100G
  • Insulin stock solution Sigma Cat. I9278-5ML, 10 mg/ml (or 25 mM) stock solution; Ready to use
  • BSA stock solution Sigma Cat. A9576-50 ml, 30% in DPBS; Ready to use
  • L-glutamine stock solution prepare on a per-use basis
  • the SFGF medium was originally designed by the inventors for use by a third party who had requested media with little or no glucose, and containing galactose as an energy source.
  • the inventors prepared such a media using other design aspects described herein, and surprisingly discovered the ability of such media to maintain the cellular homogeneity of cardiomyocytes, as well as other desirable attributes described herein
  • iCell Cardiac Maintenance Medium is also known as DS-CMM (Dialyzed Serum Cardiac Maintenance Medium).
  • DS-CMM has the same formulation as CMM, except DMEM without glucose or sodium pyruvate is the base medium utilized and dialyzed serum, galactose and sodium pyruvate are added.
  • DS-CMM is a low glucose containing medium designed to maintain the purity of cardiomyocyte cultures. This is necessary due to the purification process, and the fact that cardiomyocytes do not divide or multiply in culture, whereas the contaminating cells do.
  • This serum containing medium is designed to maintain the survival of cardiomyocyte cells grown in vitro.
  • the medium has also been designed to maintain the purity cardiomyocyte cells grown in vitro.
  • Culture medium with standard levels of glucose and normal serum induce outgrowth of the non-cardiomyocyte population. This medium maintains the cardiomyocyte purity of the culture for at least 2 weeks.
  • the medium contains:
  • Galactose Stock Solution 500 mM solution. Weigh out 4.5 g Galactose, Volume up to 50 mL DMEM-Glucose-Pyruvate.
  • Na Pyruvate Stock Solution 100 mM Solution. Weigh out 550 mg, volume up to 50 mL DMEM-Glucose-Pyruvate.
  • CMM Cardiac Maintenance Medium
  • This CMM medium is used during the differentiation process, and was used as the default (control) media for plating experiments for post cryopreserved cells.
  • This medium includes DMEM (Gibco 10567) and fetal bovine serum (Hyclone SH30396).
  • Seeding density refers to the number of cardiomyocytes added to a culture vessel. Calculated by cell counts and purity.
  • Platinum density refers to the number of cardiomyocytes attached to culture vessel after 48 hours. Calculated by cell counts and purity.
  • Platinum efficiency refers to the percentage of cardiomyocytes attached to the culture vessel (Plating density divided by seeding density) Calculated by cell counts and purity.
  • “Purity” refers to the percentage of cardiomyocytes in a given culture condition. This could be measured by quantifying the percentage of RFP positive cells in cardiomyocyte cultures derived from an iPS cell line that expresses RFP driven by a cardiomyocyte-specific promoter or by quantifying the percentage of cardiac troponin T positive cells in cardiomyocyte cultures
  • iCell cardiomyocytes used in the present examples are a purified population of human induced pluripotent stem (iPS) cell derived cardiomyocytes.
  • the iPS starting material has a genetic modification driven by a cardiomyocyte-specific promoter of the MYH6 (alpha myosin heavy chain) gene to express red fluorescent protein (RFP) and a Blasticidin resistance gene used for purification during the manufacturing process. Since cardiomyocytes do not proliferate in culture, the purity is of great importance. Any non-cardiomyocyte cells present may continue to divide in culture, so that the cardiomyocyte culture purity is constantly dropping with time.
  • MYH6 alpha myosin heavy chain
  • the cells were purified to >95% RFP positive measured by flow cytometry prior to cryopreservation and eventual use. Cells were then thawed, seeded into a culture vessel, and allowed to recover for 48 hours before being considered useable for experimentation.
  • CMM Cardiac Maintenance Medium
  • FBS Fetal Bovine Serum
  • Serum Free Glucose Free Medium maintains Cardiomyocyte Purity In Vitro—
  • cardiomyocytes When cultured in standard conditions comprising 10% fetal bovine serum and 5.5 mM Glucose, highly purified (>99%) cardiomyocytes will quickly lose purity due to the outgrowth of contaminating cells. In comparison, media prepared without glucose or serum supports equivalent cardiomyocyte survival ( FIG. 1A ) while inhibiting the outgrowth of contaminating cells, effectively maintaining the culture purity ( FIG. 1B ).
  • DS-CMM Medium maintains Cardiomyocyte Purity In Vitro—
  • cardiomyocytes may require that serum be present, but that the cardiomyocyte purity also be maintained.
  • a glucose free DMEM medium comprised of 10% dialyzed serum (10,000 MW cutoff), supplemented with galactose and sodium pyruvate (DS-CMM) maintains equivalent cardiomyocyte survival to control conditions (CMM) ( FIG. 2A ).
  • the DS-CMM medium also inhibits the outgrowth of contaminating cells, maintaining a culture with high cardiomyocyte purity ( FIG. 2B ).
  • DS-CMM maintains the purity of cardiomyocytes in culture for extended periods of time. Cardiomyocytes have been cultured in DS-CMM for up to 7 months (210 days) with little or no loss of purity as measured by cardiac troponin T flow cytometry ( FIG. 2C ).
  • DMEM Dialyzed Serum—4.95 mM Glucose
  • DS-CMM Dialyzed Serum
  • iCMM Dialyzed Serum
  • Serum Free Glucose Free Medium Produces Faster Beating Monolayers than Serum Containing Media—
  • Cardiomyocytes were pre-plated for three days in tissue culture flasks in either fetal bovine serum-containing medium or in SFGF. These cultures were then trypsinized and plated at 30,000 cells per well on 6-well multi-electrode array (MEA) plates for three days in either fetal bovine serum-containing medium or in SFGF medium. The beat rate (frequency) was measured over a 5 minute period. This data demonstrates that MEA monolayers cultured in SFGF medium beat more rapidly. The increased beating frequency reduces the incidence of beating rate oscillations that can occur in MEA cardiomyocyte beating frequency analysis, thereby increasing the stability of MEA-mediated beating frequency recordings.

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